In the life-cycle of HIV, the Rev protein regulates the temporal switch from the early regulatory to the late lytic phase. The Rev regulatory protein of HIV is a basic nuclear protein that concentrates in the nucleoli and activates the viral RNAs, by binding to a highly structured RRE (Rev Responsive Element) RNA . Our studies of Rev and RRE RNA were targeted to understand i) the minimum RRE sequence requirements for activation by Rev; ii) to explore the roles of RRE other than Rev binding in the activation process, iii) to analyze the various functional motifs of the Rev protein; and iv) to identify and characterize the function of the putative cellular factors that may bind RRE RNA, Rev, or both. To examine the nature of the cooperative interactions between the RNA binding and protein oligomerization domains of Rev, we replaced the RNA binding motif of Rev with a poly-arginine tract both in the context of Rev and the Rev/MS-C fusion protein. Functionally defective of Rev/MS-C fusion protein mutants, that had lost the presumptive RRE RNA binding motif (residues 35-50), regained the activation potential for both RRE and MS2 RNA when 9 ARGs were inserted at the deletion. From these genetic studies, we propose that poly-arginine insertion near the N-terminus of Rev promotes nucleolar targeting in a context sensitive manner, and the Rev sequence immediately flanking the 9 arginines (residues 24-35, and residues 36-50) is required for RRE RNA binding. We have cloned and characterized a HIV-1 Rev Responsive Element (RRE) RNA binding cellular factor (RBF) from a library of HeLa cDNA l coliphage recombinants. RBF bore significant sequence homology with many cellular and viral ds RNA binding proteins including the interferon inducible ds RNA activated protein kinase, PKR, and the vaccinia virus E3L protein. RBF was a competitive inhibitor of ds RNA activation of interferon induced PKR kinase in vitro and in vivo, and RBF expression in HeLa cells complemented the growth and protein synthesis defect of a vaccinia virus mutant deleted for the expression of the ds RNA binding protein, E3L.